CN105759045B - One kind is directed to aflatoxin B1Detection method - Google Patents

Abstract

The invention provides one kind to be directed to aflatoxin B₁Detection method, this method is based on direct competive ELISA technology, after first monoclonal antibody is coated with, add testing sample and catalase C100 mark aflatoxin B₁, the aflatoxin B in sample₁With the aflatoxin B of catalase mark₁It is emulative to be combined with monoclonal antibody fixed on ELISA Plate, decomposing hydrogen dioxide solution is catalyzed by catalase, the fluorescent quenching of the cadmium telluride quantum dot to mercaptopropionic acid modification is reduced, according to fluorescence intensity come aflatoxin B in judgement sample₁Content.Novelty of the invention introduces new catalase, and reaction precision is improved while cost is reduced；At the same time, present invention uses the cadmium telluride quantum dot of more sensitive novel fluorescence substrate mercaptopropionic acid modification, the luminous sensitivity of more traditional tmb substrate to be obviously improved.

Description

A kind of detection method for aflatoxin B1

technical field

The invention relates to the technical field of antigen detection, and further relates to an ELISA-based antigen detection technology, in particular to _a detection method for aflatoxin B1.

Background technique

Aflatoxin is a class of derivatives containing a dihydrofuran ring and an oxone coumarin structure, and is a secondary metabolite produced by fungi such as Aspergillus flavus and Aspergillus parasiticus. There are mainly aflatoxin B ₁ , B ₂ , G ₁ , G ₂ , M ₁ and M ₂ , among which aflatoxin B ₁ is the most widely distributed in food and is the most harmful to human health. Studies have shown that aflatoxin B ₁ has strong hepatotoxicity, carcinogenicity, mutagenicity and immunosuppressive properties to humans and animals. The International Agency for Research on Cancer (IARC) has positioned aflatoxin B ₁ as the most toxic class 1 carcinogen. Aflatoxin B ₁ often contaminates foods such as corn, peanuts, rice, nuts, and vegetable oils. Therefore, establishing a sensitive and rapid detection method is _an important technical prerequisite for effective control of aflatoxin B1.

_In the prior art, commonly used methods for detecting aflatoxin B1 include confirmatory methods and rapid screening methods. Commonly used confirmation methods include high performance liquid chromatography and liquid chromatography-mass spectrometry. Although this type of method has the characteristics of high sensitivity, it needs to rely on expensive equipment and skilled operators, and requires complicated sample pretreatment, so it cannot meet the needs of grassroots and on-site monitoring. Immunology-based rapid screening methods have been widely promoted and applied in recent years due to their advantages of high throughput, rapid detection, and low price. In particular, the enzyme-linked immunosorbent assay (ELISA) method is especially suitable for the detection of large batches of samples due to its rapidity, sensitivity, specificity, accuracy, quantification, easy operation, no need for expensive instruments and equipment, and low requirements for sample purity. Advantages, it has become the main method for rapid screening and detection _of aflatoxin B1. However, the ELISA detection methods for aflatoxin B ₁ in the prior art are generally based on horseradish peroxidase-labeled antibodies or antigens that catalyze the generation of hydroxyl radicals from hydrogen peroxide, and then oxidize the colorless chemical chromogenic substrate tetramethyl glycosides. Phenylenediamine (TMB) forms a blue product, and then the reaction is terminated with a stop solution (2M H ₂ SO ₄ ) to form a yellow solution, and the absorbance value is recorded at 450 nm. Due to the low color intensity of this type of method, the detection sensitivity is relatively low. When the content of the target substance in the sample to be tested is low, false negative results are prone to occur, which cannot meet the requirements of practical applications.

In recent years, some new signal transduction mechanisms have been reported to replace traditional ELISA signal transduction mechanisms to improve the sensitivity of ELISA, such as radioimmunoassay substrates, chemiluminescent substrates, fluorescent substrates, and resonance colloidal gold solutions. However, the construction of the luminescent system needs to fully consider the molecular biological properties of the detected object, for example, at the method level, the coating of the antibody and its binding performance with the antigen, which marker enzyme to use and the coupling method with the antibody, Selection of chromogenic substrates and specific luminescence methods; at the effect level, it is necessary to ensure the sensitivity of the chromogenic reaction and satisfy the linear relationship between the color intensity and the content of the target substance. Therefore, the ELISA detection method for aflatoxin B ₁ , especially the improvement of the method for the purpose of improving its detection sensitivity, has outstanding technical difficulties.

Contents of the invention

The present invention aims at the technical defects of the prior art, and provides a detection method for aflatoxin B ₁ to solve the low precision of the aflatoxin B ₁ ELISA detection method of the prior art.

Another technical problem to be solved by the present invention is that in the ELISA method used for the detection of aflatoxin B ₁ in the prior art, the enzyme sensitivity of the labeled antigen is low.

Another technical problem to be solved by the present invention is that in the ELISA method used in the prior art for the detection _of aflatoxin B1, the sensitivity of the chromogenic system is low.

Another technical problem to be solved by the present invention is that when the ELISA method is used to detect aflatoxin B1, the coating effect _of the anti _- aflatoxin B1 monoclonal antibody is poor during the process.

Another technical problem to be solved by the present invention is that when catalase C100 is used as the antigen labeling enzyme to perform ELISA detection on aflatoxin B ₁ , the specific process method is not clear.

Another technical problem to be solved in the present invention is when adopting catalase C100 as antigen labeling enzyme, adopting hydrogen peroxide and mercaptopropionic acid-modified cadmium telluride quantum dots as substrate to carry out ELISA detection to aflatoxin B ₁ , detection result and The linearity of the antigen concentration is not good.

Another technical problem to be solved in the present invention is that when using catalase C100 as the antigen labeling enzyme and using hydrogen peroxide and mercaptopropionic acid modified cadmium telluride quantum dots as the substrate to perform ELISA detection on aflatoxin B ₁ , the process of washing not effectively.

To achieve the above technical purpose, the present invention adopts the following technical solutions:

The invention relates to _a detection method for aflatoxin B1, which belongs to the direct competition enzyme-linked immunosorbent method, and the method is for detection _of aflatoxin B1 antigen, and the enzyme used for labeling the antigen in the method is catalase C100.

Preferably, the substrate in the method includes hydrogen peroxide and mercaptopropionic acid modified cadmium telluride quantum dots.

Preferably, the method comprises the steps of:

1) Coating anti-aflatoxin B ₁ monoclonal antibody, and then adding the sample to be tested;

2) Then add catalase C100-labeled aflatoxin B ₁ , mix and react in a dark environment at 35-39°C for 40-80 minutes, and wash;

3) Then add hydrogen peroxide solution with a concentration of 8-12 μmol/L to mix, and react in a dark environment at 35-39°C for 20-40 minutes;

4) Then add mercaptopropionic acid-modified cadmium telluride quantum dots for mixing, and react for 10-20 minutes at room temperature in a dark environment;

5) Detect the fluorescence intensity of the product of step 4).

The fluorescence intensity is used to reflect the content of aflatoxin B ₁ in the sample to be tested. In actual operation, multiple groups of aflatoxin B ₁ standard solutions with known concentrations and gradient distribution can be used to draw the fluorescence intensity-aflatoxin B1 concentration standard curve, and then use the fluorescence intensity _of the test sample to calculate the aflatoxin B1 content _of the test sample from the standard curve. The specific operation method can be selected arbitrarily according to the general technical knowledge in the technical field. For the multiple groups of aflatoxin B ₁ standard solutions with gradient distribution above, 0pg/mL, 0.05pg/mL, 0.1pg/mL, 0.2pg/mL, 0.8pg/mL, 2pg/mL, 5pg/mL can be selected respectively.

In the preferred technical scheme, in step 1), the sample to be tested is first added, so that the antigenic substance contained in the sample to be tested is combined with the antibody on the microtiter plate, and the aflatoxin B ₁ labeled with catalase C100 is added in step 2). Finally, the aflatoxin B ₁ labeled by catalase C100 competes with the antigen in the sample to be tested and binds to the solid-phase antibody; step 3) after adding hydrogen peroxide solution, it can be decomposed by catalase C100; and then step 4) add quantum Realize the luminescent effect after pointing; because the amount of catalase C100 contained on the microtiter plate is positively correlated with the luminous intensity, and is negatively correlated with the antigen content in the sample to be tested, the content of aflatoxin B ₁ in the sample to be tested is related to the luminescent intensity. Intensity is negatively correlated. In this preferred technical scheme: each reagent can be equilibrated at room temperature for more than 30 minutes before use; the amount of the sample to be tested in step 1) is preferably 30-70 μL/well, more preferably 50 μL/well; step 2 ) is preferably 30-70 μL/well, more preferably 50 μL/well; the addition of hydrogen peroxide solution in step ₃ ) is preferably 80-120 μL/well, more preferably The preferred amount is 100 μL/well; in step 4), the amount of mercaptopropionic acid-modified cadmium telluride quantum dots added is preferably 30-70 μL/well, more preferably 50 μL/well.

As preferably, the coating anti-aflatoxin B ₁ monoclonal antibody described in step 1) specifically includes the following operations:

A) Take protein G, use 0.04-0.06mol/L, pH9.4-9.8 carbonate buffer as coating solution on the microtiter plate, dilute protein G to 18-22μg/mL;

B) After removing the liquid on the microplate, wash the microplate with washing solution, then take the anti-aflatoxin B ₁ monoclonal antibody, and use the coating solution to dilute the anti-aflatoxin B ₁ monoclonal antibody in the microplate well Antibody to 0.6～1μg/mL;

C) After removing the liquid on the ELISA plate, wash the ELISA plate with washing solution, then add bovine serum albumin blocking solution, block at 35-39° C. for 1-3 hours, and then discard the blocking solution.

Further, the following optimizations can be performed: after dilution in step A), let stand at 0-8°C for 8-12 hours, and then perform step B); after dilution in step B), let stand at 35-39°C for 1-2 hours 3h, and then execute step C); step A) the coating solution is added in an amount of 80-120 μL/well, more preferably 100 μL/well; in step B, the coating solution is added in an amount of 80-120 μL/well, More preferably 100 μL/well; the concentration of bovine serum albumin in step C) is 0.3 to 0.7%, more preferably 0.5%; the enzyme label plate after discarding the blocking solution is dried at room temperature, and then dried in 2 to 0.7%. Store at 6°C.

As a preference, the aflatoxin B ₁ labeled with catase C100 in step 2) is prepared by the following method:

M) Prepare a tetrahydrofuran solution with a concentration of aflatoxin B ₁ oxime compound of 0.8-1.2 mg/mL, add N-hydroxysuccinimide to a concentration of 1.8-2.2 mg/mL, add 1-(3-dimethylamino Propyl)-3-ethylcarbodiimide hydrochloride to a concentration of 3.6-4.4mg/mL, and then react for 40-80min under dark conditions;

N) followed by solid-liquid separation, taking the supernatant to evaporate the solvent, taking the residue and dissolving it in dimethylformamide to obtain the activated product;

P) mixing the activated product described in step N) with a sodium bicarbonate solution containing catalase C100 at a concentration of 3.5-4.5 mg/mL, and reacting for 8-12 hours under dark conditions;

Q) Then dialyze to remove the free aflatoxin B ₁ oximate, that is, to obtain the aflatoxin B ₁ labeled with the catase C100.

Further, the following optimization can be performed: the solvent of the tetrahydrofuran solution described in step M) is only tetrahydrofuran; the reaction temperature in step M) is room temperature; the amount of dimethylformamide in step N) is 1/1 of the amount of tetrahydrofuran in step M). 5～3/5; the sodium bicarbonate content in the sodium bicarbonate solution described in the step P) is 0.1～0.15mol/L, more preferably 0.1～0.15mol/L; the reaction in the step P) is in the shaking condition Carry out under; step Q) described dialysis time is 66～78h, more preferably 72h; Step Q) described dialysis is carried out in the PBS solution of 0.01mol/L; Step N) described solid-liquid Separation is centrifuged at 10000r/min for 15min.

As a preference, the cadmium telluride quantum dots modified by mercaptopropionic acid in step 4) are prepared by the following method: preparing The solution of sodium hydride is the precursor solution, the pH of the precursor solution is 11-11.5, and the precursor solution is heated to 93-97°C in a water bath to obtain the mercaptopropionic acid-modified cadmium telluride quantum dots . Further preferably on this basis, a pH adjustment link may also be included, and the pH adjustment is realized by using NaOH.

Preferably, the detection of the fluorescence intensity in step 5) is realized by using a microplate reader, the excitation wavelength is 310nm, and the emission wavelength is 590nm.

Preferably on the basis of any of the above technical solutions, the washing is to rinse or soak with a washing solution, the washing solution is a PBST solution containing 0.3-0.7% (v/v) Tween-20, and the PBST solution The concentration of the PBST solution is 0.005-0.02 mol/L, and the pH of the PBST solution is 7.0-7.5.

In the above technical scheme, catalase (Catalase) is also called catalase, and the catalase C100 used in the present invention refers specifically to the catalase produced by Sigma-Aldrich Company of the United States, whose model is cat.No.C100. The catalase C100-labeled aflatoxin B ₁ refers to the substance after the catalase C100 is linked to the aflatoxin B ₁ molecule.

In the above technical schemes, the enzyme plate can be a 96-well black fluorescent enzyme plate. 96-well black fluorescent microtiter plate, anti-aflatoxin B ₁ monoclonal antibody, catalase C100, hydrogen peroxide, cadmium nitrate, mercaptopropionic acid, sodium hydride telluride and other reagents were purchased from the market.

Preferably, the washing is to add 320-360 μL/well washing solution to the enzyme-labeled wells, and wash 3-4 times with an interval of 10-30 s between each time.

As a preference, the sample to be tested is subjected to the following pretreatment before performing the detection: (1) take the pulverized sample and pass it through a 20-mesh sieve and mix it thoroughly; (2) weigh 5g of the sample and add 12.5ml of extracting solution (methanol: water = 7:3), shake vigorously for 30 minutes; (3) Centrifuge at 5000rpm for 10 minutes, filter with filter paper; (4) Dilute 1mL of the filtrate with 1mL of PBS and set aside.

The present invention adopts enzyme-linked immunosorbent assay method to detect. The determination principle of the ELISA kit for the detection of aflatoxin B ₁ : the aflatoxin B _{1 in the sample competes with the catalase-labeled aflatoxin B 1 and} the anti-aflatoxin B ₁ monoclonal immobilized on the ELISA plate The cloned antibody binds, catalyzes the decomposition of hydrogen peroxide by catalase, reduces the fluorescence quenching of mercaptopropionic acid-modified cadmium telluride quantum dots, and judges the content of aflatoxin B ₁ in the sample according to the fluorescence intensity. If the content of aflatoxin B ₁ in the sample is low, the fluorescence intensity is high; otherwise, the fluorescence intensity is low. That is, the level of fluorescence intensity is inversely proportional to the content of aflatoxin B ₁ in the standard or sample. The method can be directly used to detect aflatoxin B ₁ in corn and corn products. The detection method of the kit of the invention is easy to operate, sensitive, accurate and fast, and is suitable for the detection of large batches of samples.

Adopting the technical solution of the present invention has the following beneficial effects:

1. The method of the present invention uses a more sensitive new catalase to replace the horseradish peroxidase in the traditional ELISA method, which can greatly save the cost.

2. The method of the present invention uses a more sensitive novel fluorescent substrate (cadmium telluride quantum dot modified with mercaptopropionic acid) to replace the chemical chromogenic substrate in the traditional ELISA method, which can greatly improve its detection sensitivity. Compared with the traditional ELISA Improved at least 2-3 orders of magnitude.

Description of drawings

Fig. 1 is a principle comparison chart of the detection method of the present invention and a conventional detection method using horseradish peroxidase as an antibody marker enzyme and TMB as a substrate.

Fig. 2 is a standard curve diagram of percent fluorescence ratio-aflatoxin B ₁ concentration in Example 1 of the present invention.

Fig. 3 is the percent absorbance-aflatoxin B ₁ concentration standard curve diagram in Example 2 of the present invention.

detailed description

Specific embodiments of the present invention will be described in detail below. In order to avoid too many unnecessary details, well-known structures or functions will not be described in detail in the following embodiments.

Approximate language used in the following examples is for quantitative representations, indicating that certain variations in quantities are permissible without altering essential function. Accordingly, values modified by language such as "about", "approximately" and the like are not limited to the exact value itself. In some embodiments, "about" means that the corrected value is allowed to vary within the range of plus or minus ten percent (10%), for example, "about 100" means any value between 90 and 110. Furthermore, in the expression "about the first value to the second value", both values of the first value and the second value are corrected approximately at the same time. In some cases, the language of approximation may relate to the precision of the measuring instrument.

Unless defined otherwise, technical and scientific terms used in the following examples have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The test reagent consumables used in the following examples, if no special instructions, are conventional biochemical reagents; the experimental methods, if no special instructions, are conventional methods; the quantitative tests in the following examples are all provided with three repeated experiments, The results are average values; % in the following examples, unless otherwise specified, are mass percentages.

In the following examples, the 96 _- well black fluorescent microtiter plate was purchased from Corning Corporation _of the United States _; from Wuxi Zhongdeboer Biotechnology Co., Ltd.; the catalase (cat.No.C100) and substrate solution A hydrogen peroxide were purchased from Sigma-Aldrich Company of the United States (35%, cat.No.349887). The catalase C100 refers to catalase (cat. No. C100).

Embodiment ₁ (a test kit is prepared based on the novel fluorescent ELISA detection method of aflatoxin B1 _of the present invention, and it is used for detecting the aflatoxin B1 residue in corn and corn products)

Preparation and detection method of a novel fluorescent ELISA kit for detecting aflatoxin B ₁ , including a 96-well black fluorescent microtiter plate coated with an anti-aflatoxin B ₁ monoclonal antibody, aflatoxin B ₁ standard, and catalase labeling Aflatoxin B ₁ working solution, substrate solution A hydrogen peroxide, fluorescent substrate solution B mercaptopropionic acid modified cadmium telluride quantum dots and concentrated washing solution.

Describe in detail below the preparation _of the ELISA kit that detects aflatoxin B1 in the present invention,

The 96-well black fluorescent microtiter plate was purchased from Corning Company in the United States; the anti-aflatoxin B ₁ monoclonal antibody was purchased from Wuxi Zhongdeboer Biotechnology Co., Ltd.; catalase (cat.No.C100) and substrate solution The hydrogen peroxide described in A was purchased from Sigma-Aldrich Company of the United States (35%, cat. No. 349887).

The catalase-labeled aflatoxin B ₁ was obtained by dissolving 0.5 mg of aflatoxin B ₁ oximate in 500 μL of anhydrous tetrahydrofuran, adding 1 mg of N-hydroxysuccinimide and 2.0 mg of 1-(3 -Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, react at room temperature in the dark for 60 minutes; centrifuge at 10,000 r/min for 15 minutes, discard the precipitate, dry the supernatant, evaporate tetrahydrofuran, and dissolve the residue in 200 μL dimethyl In formamide, the activated product was obtained; the activated product was slowly added dropwise to the catalase solution (4mg, dissolved in 1mL 0.13mol/L NaHCO ₃ solution), shaken vigorously at room temperature in the dark, and the reaction product was dissolved in 0.01mol/L PBS The solution was dialyzed for 72 hours to remove free aflatoxin B ₁ oximate; after the dialysis, the sample was freeze-dried to obtain catalase-labeled aflatoxin B ₁ , which was aliquoted and stored at -20°C.

The cadmium telluride quantum dots modified by the fluorescent substrate liquid B mercaptopropionic acid are obtained in the following manner: take a freshly configured sodium hydride telluride solution and add it to the cadmium nitrate solution, and add 1 mol/L sodium hydroxide solution to adjust the pH value to 11.2. Add mercaptopropionic acid solution to the mixed solution as a stabilizer, and heat the formed cadmium telluride precursor solution to 95°C in a water bath. The concentration of adding each solution in the precursor solution is that the final concentration of cadmium nitrate solution is 10mmol/L, the final concentration of mercaptopropionic acid is 24mmol/L, and the final concentration of sodium hydride telluride is 5mmol/L.

Preparation of the 96-well black fluorescent microplate plate coated with the anti-aflatoxin B ₁ monoclonal antibody:

Use 0.05mol/L carbonate (CBS) buffer solution with pH 9.6 as the coating solution, dilute protein G (purchased from Shanghai Yanhui Biotechnology Co., Ltd., cat.No.PRO-402) to 20μg/mL, 100μL /well, place overnight at 4°C, take out the ELISA plate and shake off the liquid in the plate, wash the plate 3 times with 340μL/well of diluted concentrated washing solution, 30s/time; then dilute the anti-aflatoxin B ₁ monoclonal antibody 0.8 μg/mL, 100 μL/well, place at 37°C for 2 hours, take out the plate and shake off the liquid in the plate, wash the plate 3 times with 340 μL/well of concentrated washing solution after dilution, 30 s/time; finally add 0.5% bovine Block with serum albumin (BSA, purchased from Sigma-Aldrich, USA, cat. No. A4737), 340 μL/well, place at 37°C for 2 hours, discard the blocking solution, and place the plate in a constant temperature room (25°C) after being patted dry. Dry in the air; after passing the sampling inspection, vacuum-seal the microplate and store it at 4°C. The prepared concentrations of the aflatoxin B ₁ standard were 0pg/mL, 0.05pg/mL, 0.1pg/mL, 0.2pg/mL, 0.8pg/mL, 2pg/mL, 5pg/mL.

Preparation of the catalase-labeled aflatoxin B ₁ working solution: obtained by coupling aflatoxin B ₁ with catalase, diluted with PBS (0.01M, pH7.4) to 1:1280.

Preparation of the substrate liquid A hydrogen peroxide: dilute to 10 μmol/L with PBS (0.01 mol/L, pH 7.4). The preparation of the mercaptopropionic acid-modified cadmium telluride quantum dot fluorescent substrate solution: method, dilute to 1:400 with PBS (0.01mol/L, pH7.4).

The concentrated washing liquid is a 10-fold concentrated washing liquid, which contains 0.5% Tween-20, 0.01 mol/L PBST, and a pH range of 7.0-7.5.

Based on the reagents prepared above, the ELISA kit for detecting aflatoxin B ₁ of the present invention includes the following materials:

(1) 96-well ELISA plate × 1 piece (coated with monoclonal antibody against aflatoxin B ₁ );

(2) Standard solution × 7 bottles: (1mL/bottle) 0pg/mL, 0.05pg/mL, 0.1pg/mL, 0.2pg/mL, 0.8pg/mL, 2pg/mL, 5pg/mL;

(3) Catalase-labeled aflatoxin B ₁ working solution 8mL;

(4) Substrate solution A 7mL;

(5) Fluorescence substrate solution B 7mL;

(6) Stop solution 7mL;

(7) 20 mL of 10× concentrated washing solution.

Precautions when using this kit:

(1) Reagents and samples taken out of the refrigerator should be warmed to 20-25°C;

(2) If the plate wells are dry during the plate washing process, the standard curve will not be linear and the repeatability will be poor. Therefore, the next step should be performed immediately after washing the plate and drying it;

(3) Shake well before adding each reagent;

(4) The substrate solution A is hydrogen peroxide with a concentration of 35%, avoiding direct contact with the skin;

(5) Do not use the kit with the expiry date; do not use any reagent in the kit that has passed the expiry date. Doping with the kit that has passed the expiry date will cause a decrease in sensitivity; do not exchange the kits with different batch numbers reagents;

(6) Storage conditions: Store the kit at 2-8°C, do not freeze, put the unused microplate microplate into a ziplock bag and reseal it. Substrate A and Fluorescent Substrate B are sensitive to light, so avoid direct exposure to light;

(7) The optimal reaction temperature of the kit is 25°C, if the temperature is too high or too low, the absorbance value and sensitivity of the test will change.

When the kit of the present invention is used to detect the residual amount _of aflatoxin B1 in corn and corn products, it is implemented through the following steps: sample pretreatment, detection with the kit of the present invention, and analysis of the results.

(1) Sample pretreatment

Take the pulverized sample and pass it through a 20-mesh sieve, and mix thoroughly; weigh 5g sample, add 12.5mL extract solution (methanol:water=7:3), shake vigorously for 30min; centrifuge at 5000rpm for 10min, filter with filter paper; take 1mL filtrate for Dilute with 1mL PBS and set aside.

(2) detect aflatoxin B ₁ residual amount in the above-mentioned sample with kit of the present invention

Take the microtiter plate coated with anti-aflatoxin B ₁ monoclonal antibody, add standard substance/sample 50 μL/well to the corresponding microwell; add catalase-labeled aflatoxin B ₁ working solution, 50 μL/well, use The cover film and the cover plate were reacted for 45 minutes at a room temperature of 37°C in a light-proof environment; carefully uncover the cover film, dry the liquid in the well, and wash fully with 340 μL/well of washing working solution for 4 to 5 times, with an interval of 10 seconds between each time , pat dry with absorbent paper; add substrate solution A hydrogen peroxide (10μmol/L) diluent, 100μL/well, shake and mix gently, cover the plate with a cover film and place it in a light-proof environment at 37°C for 30min; add fluorescence Substrate solution B mercaptopropionic acid-modified cadmium telluride quantum dot dilution 50 μL/well, shake gently to mix, cover the plate with a cover film and place it in a light-proof environment at 25°C for 15 minutes, set the fluorescent microplate reader ( The American Thermo Varioskan Flash full-wavelength multifunctional microplate reader) detects at the excitation wavelength of 310nm and the emission wavelength of 590nm, and measures the fluorescence value of each well (please read the data within 5min); Concentration logarithmic value draws standard curve, the content of aflatoxin B ₁ in the reference standard curve calculation sample.

(3) Analysis results

Use the 7 aflatoxin B ₁ standard concentrations 0pg/mL, 0.05pg/mL, 0.1pg/mL, 0.2pg/mL, 0.8pg/mL, 2pg/mL, 5pg/mL in the kit prepared above. Fluorescence intensity was detected at an excitation wavelength of 310 nm and an emission wavelength of 590 nm.

For the calculation of the percent fluorescence rate, the percent fluorescence rate of the standard or sample is equal to the average value (double holes) of the percent fluorescence intensity values of the standard or sample divided by the fluorescence intensity value of the first standard (0 standard), and then Multiply by 100%, that is, percent fluorescence rate (%)=F/F ₀ ×100%, where F is the average fluorescence intensity value of the standard solution or sample solution, and F ₀ is the average fluorescence intensity value of the 0pg/mL standard solution.

Draw the standard curve with the percent fluorescence rate of the standard as the ordinate and the semi-logarithm of the aflatoxin B ₁ standard concentration (pg/mL) as the abscissa, and obtain the linear equation. The standard curve is y=-17.3286ln(x)+38.625, R ² =0.9984, see Figure 2. The _IC50 of this method is defined as the concentration of aflatoxin B1 corresponding to _a percent light ratio of 50%. The IC ₅₀ calculated by this standard curve was 0.518 pg/mL. When performing actual sample detection, substitute the percent fluorescence rate (F/F ₀ ×100%) value of the sample into the standard curve, read the concentration of the corresponding sample from the standard curve, and multiply it by the corresponding dilution factor. The actual concentration of aflatoxin B ₁ in the sample.

Embodiment 2 (using horseradish peroxidase as antibody labeling enzyme, using TMB as the aflatoxin B ₁ direct competition ELISA detection method of chromogenic substrate)

When the traditional ELISA kit is used to detect the residual amount _of aflatoxin B1 in corn and corn products, it is implemented through the following steps: sample pretreatment, detection with the kit of the present invention, and analysis of the results.

(1) Sample pretreatment

Take the pulverized sample and pass it through a 20-mesh sieve, and mix thoroughly; weigh 5g sample, add 12.5mL extract solution (methanol:water=7:3), shake vigorously for 30min; centrifuge at 5000rpm for 10min, filter with filter paper; take 1mL filtrate for Dilute with 1mL PBS and set aside.

(2) Detect aflatoxin B ₁ residues in the above samples with a traditional ELISA kit

Take the ELISA plate coated with anti-aflatoxin B ₁ monoclonal antibody, add standard substance/sample 50 μL/well to the corresponding microwell; add horseradish peroxidase-labeled aflatoxin B ₁ working solution, 50 μL Cover the plate with the cover film and react in a light-proof environment at room temperature 37°C for 45 minutes; carefully uncover the cover film, dry the liquid in the well, and wash fully with 340 μL/well of washing working solution for 4 to 5 times. Pat dry with absorbent paper at intervals of 10 seconds each time; add TMB chromogenic solution, 100 μL/well, shake and mix gently, cover the plate with a cover film and place it in a dark environment at 37°C for 15 minutes; add stop solution 50 μL/well , shake and mix gently, set the microplate reader to detect at 450nm, and measure the absorbance value of each well (please read the data within 5 minutes); compare the absorbance value of the sample to be tested with the standard, and quantitatively analyze the absorbance of the sample to be tested. Residues _of aflatoxin B1.

(3) Analysis results

Use 6 aflatoxin B ₁ standard concentrations of 0ng/mL, 0.0195ng/mL, 0.0391ng/mL, 0.1563ng/mL, 0.3125ng/mL, 1.25ng/mL in the traditional ELISA kit. Absorbance values were measured at 450 nm.

For the calculation of percent absorbance, the percent absorbance of a standard or sample is equal to the average (double well) of the percent absorbance of the standard or sample divided by the absorbance of the first standard (0 standard), and then multiplied by 100%, that is, percent absorbance value (%)=B/B ₀ ×100% where B is the average absorbance value of the standard solution or sample solution, and B ₀ is the average absorbance value of the 0ng/mL standard solution.

Draw the standard curve with the percent absorbance of the standard substance as the ordinate, and the semi-logarithm of the concentration of aflatoxin B ₁ standard substance (ng/mL) as the abscissa, and obtain the linear equation. The standard curve is y=-17.214ln(x)+17.7497, R ² =0.9979, see Figure 3. The _IC50 of this method is defined as the concentration of aflatoxin B1 corresponding to _a percent absorbance of 50%. The IC ₅₀ calculated by this standard curve was 0.153 ng/mL. When performing actual sample detection, substitute the percent fluorescence rate (B/B ₀ × 100%) of the sample into the standard curve, read the concentration of the corresponding sample from the standard curve, and multiply it by the corresponding dilution factor. The actual concentration of aflatoxin B ₁ in the sample.

By contrasting Example 1 and Example 2 above, it can be found that the sensitivity of the novel ELISA method of the present invention can reach 295 times [(153pg/mL)/(0.518pg/mL)] of the classic ELISA method, and it also proves that the novel ELISA method of the present invention The ELISA method can be adapted to detect with high sensitivity any substance that can be detected by conventional ELISA methods.

Example 3

The invention relates to _a detection method for aflatoxin B1, which belongs to the direct competition enzyme-linked immunosorbent method. The method is for the detection _of aflatoxin B1 antigen, and the enzyme used for labeling the antigen in the method is catalase C100.

On the basis of the above technical solutions, the following conditions are met:

The substrate in the method includes hydrogen peroxide and mercaptopropionic acid modified cadmium telluride quantum dots.

The specific detection method includes the following steps:

1) Coating anti-aflatoxin B ₁ monoclonal antibody, and then adding the sample to be tested;

2) Then add catalase C100-labeled aflatoxin B ₁ , mix and react in a dark environment at 35°C for 40 minutes, and wash;

3) Then add hydrogen peroxide solution with a concentration of 8 μmol/L to mix, and react for 20 minutes at 35°C in a dark environment;

4) Then add mercaptopropionic acid-modified cadmium telluride quantum dots to mix, and react for 10 minutes at 35°C in a dark environment;

5) Detect the fluorescence intensity of the product of step 4).

The coating anti-aflatoxin B ₁ monoclonal antibody described in step 1) specifically includes the following operations:

A) Take protein G, use 0.04mol/L, pH9.4 carbonate buffer as the coating solution on the microtiter plate, dilute protein G to 18 μg/mL, and let it stand at 0°C for 8 hours;

B) After removing the liquid on the microplate, wash the microplate with washing solution, then take the anti-aflatoxin B ₁ monoclonal antibody, and use the coating solution to dilute the anti-aflatoxin B ₁ monoclonal antibody in the microplate well Antibody to 0.6μg/mL, stand at 35°C for 1h;

C) After removing the liquid on the ELISA plate, wash the ELISA plate with washing solution, then add bovine serum albumin blocking solution, block at 35° C. for 1 hour, and then discard the blocking solution.

Step 2) The aflatoxin B ₁ labeled with catalase C100 is prepared by the following method:

M) Prepare a tetrahydrofuran solution with aflatoxin B ₁ oxime concentration of 0.8 mg/mL, add N-hydroxysuccinimide to a concentration of 1.8 mg/mL, add 1-(3-dimethylaminopropyl)- 3-Ethylcarbodiimide hydrochloride to a concentration of 3.6mg/mL, and then reacted for 40min under dark conditions;

N) then centrifuge at 10000r/min for 15min to carry out solid-liquid separation, take the supernatant to evaporate the solvent, take the residue and dissolve it in dimethylformamide to obtain the activated product;

P) Mix the activated product described in step N) with a sodium bicarbonate solution containing catalase C100 at a concentration of 3.5 mg/mL, and react for 8 hours under dark conditions;

Q) Then dialyze to remove the free aflatoxin B ₁ oximate, that is, to obtain the aflatoxin B ₁ labeled with the catase C100.

Step 4) The cadmium telluride quantum dots modified by mercaptopropionic acid are prepared by the following method: preparing a solution containing 8mmol/L cadmium nitrate, 20mmol/L mercaptopropionic acid, and 3mmol/L sodium hydride telluride is the precursor solution , the pH of the precursor solution is 11, and the precursor solution is heated to 93° C. in a water bath to obtain the mercaptopropionic acid-modified cadmium telluride quantum dots.

The detection of the fluorescence intensity in step 5) is realized by using a microplate reader, the excitation wavelength is 310nm, and the emission wavelength is 590nm.

Described washing is to utilize washing solution to rinse or soak, and described washing solution is the PBST solution that contains 0.3% (v/v) Tween-20, and the concentration of described PBST solution is 0.005mol/L, and the pH of described PBST solution is 7.0.

Example 4

The invention relates to _a detection method for aflatoxin B1, which belongs to the direct competition enzyme-linked immunosorbent method, and the method is for detection _of aflatoxin B1 antigen, and the enzyme used for labeling the antigen in the method is catalase C100.

On the basis of the above technical solutions, the following conditions are met:

The substrate in the method includes hydrogen peroxide and mercaptopropionic acid modified cadmium telluride quantum dots.

The specific detection method includes the following steps:

1) Coating anti-aflatoxin B ₁ monoclonal antibody, and then adding the sample to be tested;

2) Then add catalase C100-labeled aflatoxin B ₁ , mix and react for 80 minutes at 39°C in a dark environment, and wash;

3) Then add hydrogen peroxide solution with a concentration of 12 μmol/L to mix, and react for 40 minutes at 39°C in a dark environment;

4) Then add mercaptopropionic acid-modified cadmium telluride quantum dots to mix, and react for 20 minutes at 39°C in a light-proof environment;

5) Detect the fluorescence intensity of the product of step 4).

The coating anti-aflatoxin B ₁ monoclonal antibody described in step 1) specifically includes the following operations:

A) Take protein G, use 0.06mol/L, pH9.8 carbonate buffer as the coating solution on the microtiter plate, dilute protein G to 22 μg/mL, and let it stand at 8°C for 12 hours;

B) After removing the liquid on the microplate, wash the microplate with washing solution, then take the anti-aflatoxin B ₁ monoclonal antibody, and use the coating solution to dilute the anti-aflatoxin B ₁ monoclonal antibody in the microplate well Antibody to 1 μg/mL, let stand at 39°C for 3 hours;

C) After removing the liquid on the ELISA plate, wash the ELISA plate with washing solution, then add bovine serum albumin blocking solution, block at 39° C. for 3 hours, and then discard the blocking solution.

Step 2) The aflatoxin B ₁ labeled with catalase C100 is prepared by the following method:

M) Prepare a tetrahydrofuran solution with aflatoxin B ₁ oxime concentration of 1.2 mg/mL, add N-hydroxysuccinimide to a concentration of 2.2 mg/mL, add 1-(3-dimethylaminopropyl)- 3-Ethylcarbodiimide hydrochloride to a concentration of 4.4mg/mL, and then reacted for 80min under dark conditions;

N) then centrifuge at 10000r/min for 15min to carry out solid-liquid separation, take the supernatant to evaporate the solvent, take the residue and dissolve it in dimethylformamide to obtain the activated product;

P) Mix the activated product described in step N) with a sodium bicarbonate solution containing catalase C100 at a concentration of 4.5 mg/mL, and react for 12 hours under dark conditions;

Q) Then dialyze to remove the free aflatoxin B ₁ oximate, that is, to obtain the aflatoxin B ₁ labeled with the catase C100.

Step 4) The cadmium telluride quantum dots modified by mercaptopropionic acid are prepared by the following method: preparing a solution containing 12mmol/L cadmium nitrate, 28mmol/L mercaptopropionic acid, and 7mmol/L sodium hydride telluride is the precursor solution , the pH of the precursor solution is 11.5, and the precursor solution is heated to 97° C. in a water bath to obtain the mercaptopropionic acid-modified cadmium telluride quantum dots.

The detection of the fluorescence intensity in step 5) is realized by using a microplate reader, the excitation wavelength is 310nm, and the emission wavelength is 590nm.

Described washing is to utilize washing solution to flush or soak, and described washing solution is the PBST solution that contains 0.7% (v/v) Tween-20, and the concentration of described PBST solution is 0.02mol/L, and the pH of described PBST solution is 7.5.

Example 5

The invention relates to _a detection method for aflatoxin B1, which belongs to the direct competition enzyme-linked immunosorbent method, and the method is for detection _of aflatoxin B1 antigen, and the enzyme used for labeling the antigen in the method is catalase C100.

On the basis of the above technical solutions, the following conditions are met:

The substrate in the method includes hydrogen peroxide and mercaptopropionic acid modified cadmium telluride quantum dots.

The specific detection method includes the following steps:

1) Coating anti-aflatoxin B ₁ monoclonal antibody, and then adding the sample to be tested;

2) Then add catalase C100-labeled aflatoxin B ₁ , mix and react for 60 minutes at 37°C in a dark environment, and wash;

3) Then add hydrogen peroxide solution with a concentration of 10 μmol/L to mix, and react for 60 minutes at 37°C in a dark environment;

4) Then add mercaptopropionic acid-modified cadmium telluride quantum dots to mix, and react for 15 minutes at 37°C in a light-proof environment;

5) Detect the fluorescence intensity of the product of step 4).

Example 6

_A detection method for aflatoxin B1, the method belongs to direct competitive enzyme-linked immunosorbent assay, the method is for the detection _of aflatoxin B1 antigen, the enzyme used to label the antigen in the method is catalase C100, the method The substrate in the method includes hydrogen peroxide and mercaptopropionic acid modified cadmium telluride quantum dots.

Example 7

The invention relates to _a detection method for aflatoxin B1, which belongs to the direct competition enzyme-linked immunosorbent method, and the method is for detection _of aflatoxin B1 antigen, and the enzyme used for labeling the antigen in the method is catalase C100.

The embodiments of the present invention have been described in detail above, but the content is only a preferred embodiment of the present invention, and is not intended to limit the present invention. All modifications, equivalent replacements and improvements made within the application scope of the present invention shall be included in the protection scope of the present invention.

Claims (9)

1. one kind is directed to aflatoxin B₁Detection method, this method belongs to Direct cELISA, and this method is to be directed to Aflatoxin B₁The detection of antigen, the enzyme that labelled antigen is used in this method are catalase C100, and substrate includes double in this method Oxygen water and the cadmium telluride quantum dot of mercaptopropionic acid modification.

2. according to claim 1 be directed to aflatoxin B₁Detection method, it is characterised in that comprise the following steps：

1) it is coated with aspergillus flavus resisting toxin B₁Monoclonal antibody, then add testing sample；

2) aflatoxin B of catalase C100 marks is then added₁, mix after in 35~39 DEG C of light protected environments reaction 40~ 80min, washing；

3) the hydrogen peroxide solution mixing that concentration is 8~12 μm of ol/L is then added, react 20 in 35~39 DEG C of light protected environments~ 40min；

4) the cadmium telluride quantum dot mixing of mercaptopropionic acid modification is then added, 10~20min is reacted in room temperature light protected environment；

5) detecting step 4) product fluorescence intensity.

3. according to claim 2 be directed to aflatoxin B₁Detection method, it is characterised in that be coated with described in step 1) Aspergillus flavus resisting toxin B₁Monoclonal antibody specifically includes following operation：

A) take Protein G, on ELISA Plate using 0.04~0.06mol/L, pH9.4~9.8 carbonate buffer solution as coating buffer, Diluted protein G to 18~22 μ g/mL；

B cleaning solution washing ELISA Plate is utilized after) removing the liquid on ELISA Plate, then takes aspergillus flavus resisting toxin B₁Monoclonal antibody, Aspergillus flavus resisting toxin B is diluted in enzyme mark hole using the coating buffer₁Monoclonal antibody is to 0.6~1 μ g/mL；

C cleaning solution washing ELISA Plate is utilized after) removing the liquid on ELISA Plate, bovine serum albumin(BSA) confining liquid is added, in 35 ~39 DEG C of 1~3h of closing, then discard confining liquid.

4. according to claim 3 be directed to aflatoxin B₁Detection method, it is characterised in that step A) in dilute after, 8~12h is stood under the conditions of 0~8 DEG C, then performs step B)；Step B) in dilute after, under the conditions of 35~39 DEG C stand 1~ 3h, then perform step C).

5. according to claim 2 be directed to aflatoxin B₁Detection method, it is characterised in that the step 2) catalase The aflatoxin B of C100 marks₁It is prepared by the following method：

M aflatoxin B) is prepared₁Oxime compounds concentration is 0.8~1.2mg/mL tetrahydrofuran solution, adds N- maloyls Imines to concentration is 1.8~2.2mg/mL, adds 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides to concentration and is 3.6~4.4mg/mL, and 40~80min is reacted under the conditions of lucifuge；

N) then separation of solid and liquid, takes supernatant solvent flashing, takes residue to be dissolved in dimethylformamide, that is, obtains activation production Thing；

P) by step N) activation products mix with the sodium bicarbonate solution that the concentration of C100 containing catalase is 3.5~4.5mg/mL, 8~12h is reacted under the conditions of lucifuge；

Q) then dialysis removes free aflatoxin B₁Oxime compounds, that is, obtain the aflatoxin of the catalase C100 marks B₁。

6. according to claim 5 be directed to aflatoxin B₁Detection method, it is characterised in that step N) described in solid-liquid Separation is 10000r/min centrifugations 15min.

7. according to claim 2 be directed to aflatoxin B₁Detection method, it is characterised in that the step 4) sulfydryl third The cadmium telluride quantum dot of acid modification is prepared by the following method：Prepare containing 8~12mmol/L cadmium nitrates, 20~ The solution that 28mmol/L mercaptopropionic acids, 3~7mmol/L hydrogen tellurides are received is precursor solution, the pH of the precursor solution for 11~ 11.5, by the precursor solution heating water bath to 93~97 DEG C, that is, obtain the cadmium telluride quantum dot of the mercaptopropionic acid modification.

8. according to claim 2 be directed to aflatoxin B₁Detection method, it is characterised in that fluorescence intensity in step 5) Detection utilize ELIASA realize, excitation wavelength 310nm, launch wavelength 590nm.

9. it is directed to aflatoxin B according to any one of claim 2~8₁Detection method, it is characterised in that it is described to wash Washing is rinsed or is soaked using cleaning solution, and the cleaning solution is the PBST solution containing 0.3~0.7% (v/v) Tween-20, institute The concentration for stating PBST solution is 0.005~0.02mol/L, and the pH of the PBST solution is 7.0~7.5.